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Infectious bovine rhinotracheitis virus strain and preparation method and application thereof

A rhinotracheitis virus, infectious technology, applied in the field of infectious bovine rhinotracheitis virus, can solve the problems of high sensitivity, time-consuming, and false positives, and achieve the effects of high sensitivity, simple operation, and high accuracy

Active Publication Date: 2015-04-01
湖南普简生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods all have shortcomings in varying degrees, or the detection sensitivity is not high, or time-consuming, laborious, or have false positives; and PCR is simple and easy to operate and has high sensitivity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Using PCR to detect and identify infectious bovine rhinotracheitis virus, add 0.01mol / L PBS to the bovine nasal cavity cotton swab, shake and wash thoroughly, take 200 μL of the flushing solution and use the Viral Nucleic AcidExtraction Kit II DNA extraction kit to extract DNA , IBRV PCR method and IBRV fluorescent PCR method for detection, PCR primers are:

[0019] gB-1: 5'-TAC GAC TCG TTC GCG CTC TC-3';

[0020] gB-2: 5'-GGT ACG TCT CCA AGC TGC CC-3';

[0021] The size of the amplified fragment is 478bp. Add 12.5 μL of 2×Master Mix (product of TAKARA company), 5 μL of sample nucleic acid, and ddH2O to 25 μL in the 25 μL PCR reaction system. The amplification conditions are: 95°C pre-denaturation for 5min, 95°C for 1min, 65°C for 45s, 72°C for 45s, 10 cycles; 95°C for 1min, 54°C for 45s, 72°C for 45s, 15 cycles; 95°C for 1min, 60°C for 45s , 72°C for 45s, 10 cycles, and finally 72°C for 10min. After the PCR product was electrophoresed on 1.6% agarose gel,...

Embodiment 2

[0022] Embodiment two: detect and identify infectious bovine rhinotracheitis virus with fluorescent PCR, fluorescent PCR primers are:

[0023] gB-F: 5'-TGT GGA CCT AAA CCT CAC GGT 3';

[0024] gB-R: 5'-GTA GTC GAG CAG ACC CGT GTC-3';

[0025] TaqMan probe: 5'-FAM-AGG ACC GCG AGT TCT TGC CGC-TAMRA-3'.

[0026] Using TaqMan fluorescent PCR kit (product of TAKARA company), add 12.5 μL of 2×Master Mix, 5 μL of sample nucleic acid, and ddH2O to 25 μL in the 25 μL reaction system. Amplification conditions were: 95°C pre-denaturation for 5 minutes, 95°C for 15s, 60°C for 45s (signal collection), 45 cycles, and observed the amplification curve and Ct value. At the same time, the TaqMan fluorescent RT-PCR kit of BVDV was used to detect BVDV, the bovine bluetongue virus (BTV) nested RT-PCR method was used for detection, and the bovine leukemia virus (BLV) nested RT-PCR method was used for detection. Foot-and-mouth disease virus (FMDV) was detected by fluorescent RT-PCR. All reaction...

Embodiment 3

[0027] Example 3: Isolation of virus, IBRV PCR and fluorescent PCR were taken positive, after the cavity cotton swab washing solution was mixed, double-resistant penicillin 1000IU / mL and streptomycin 1000μg / mL were added, and then acted at 37°C for 2h, centrifuged at 2000g for 10min, and taken The supernatant was inoculated on a 96-well plate overgrown with a single layer of MDBK cells, 100 μL / well, plus 6 wells. Adsorb at 37°C for 1 hour, discard the supernatant, add maintenance solution (MEM containing 200 IU / mL penicillin, 200 μg / mL streptomycin and 2% calf serum), culture at 5% CO2 at 37°C, and observe the cytopathic changes (CPE ), when 70%-80% of the cells appear CPE to collect the poison, if the cells do not appear CPE, after 5 days blind passage, the isolate is passed to the fifth generation. At 48h of the first generation, it was found that the dead cells increased significantly. By the 4th day, the cells basically fell off and died, while the control cells grew well;...

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Abstract

The invention discloses an infectious bovine rhinotracheitis virus strain and a preparation method and application thereof. A wild strain of infectious bovine rhinotracheitis virus is found. The preparation method comprises the following steps of: firstly, identifying a sample by a PCR (Polymerase Chain Reaction) and a fluorescence PCR; then adding bovine serum which is detected to be free of IBRV (Infectious Bovine Rhinotracheitis Virus) to MDBK (Madindarby Bovine Kidney) cells by means of a culture medium, cultivating for a certain period of time so as to grow single-layer cells; after inoculating the single-layer cells with the processed sample, observing the cytopathic effect; after taking a pathological cell sample and identifying the pathological cell to be positive by the PCR, measuring TCID50 (Tissue Culture Infective Dose) as 106.2, and carrying out the virus neutralization test, wherein the TCID is 27; then inoculating the sample into the single layer cells; and when 70 percent to 80 percent of cells undergo the cytopathic effect and storing the cells into a refrigerator at a temperature of -40 DEG C. The infectious bovine rhinotracheitis virus strain and the preparation method disclosed by the invention can be applied to preparation of an engineered vaccine for preventing the infectious bovine rhinotracheitis.

Description

technical field [0001] The invention relates to an infectious bovine rhinotracheitis virus, in particular to a cell engineering vaccine for preventing bovine infectious bovine rhinotracheitis disease. Background technique [0002] Infectious bovine rhinotracheitis is an important viral infectious disease that seriously endangers the cattle industry in the world. The main pathogen of the disease is infectious bovine rhinotracheitis virus, and the prevention and control of the disease mainly uses viral drugs and chemical drugs, but due to the shortcomings of sensitivity and drug residues, it is urgent to develop a new type of vaccine. [0003] Bovine infectious rhinotracheitis virus has all the morphological characteristics of members of the herpesviridae family. The diameter of mature virions with envelopes is about 150nm to 220nm. The virus is spherical, and the nucleocapsid is a stereosymmetric icosahedron with 162 Capsomer, surrounded by a layer of lipid-containing capsul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12Q1/70C12Q1/68A61K39/265A61P31/22C12R1/93
Inventor 金业
Owner 湖南普简生物科技有限公司
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